WO2021163455A1 - Technologie de revêtement de membrane cellulaire évolutive et facile à la fois pour des particules chargées positivement et négativement - Google Patents

Technologie de revêtement de membrane cellulaire évolutive et facile à la fois pour des particules chargées positivement et négativement Download PDF

Info

Publication number
WO2021163455A1
WO2021163455A1 PCT/US2021/017823 US2021017823W WO2021163455A1 WO 2021163455 A1 WO2021163455 A1 WO 2021163455A1 US 2021017823 W US2021017823 W US 2021017823W WO 2021163455 A1 WO2021163455 A1 WO 2021163455A1
Authority
WO
WIPO (PCT)
Prior art keywords
cell membrane
core particle
cpg
msn
fnc
Prior art date
Application number
PCT/US2021/017823
Other languages
English (en)
Inventor
Kam W. Leong
Hanze HU
Dan SHAO
Chao Yang
Original Assignee
The Trustees Of Columbia University In The City Of New York
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The Trustees Of Columbia University In The City Of New York filed Critical The Trustees Of Columbia University In The City Of New York
Publication of WO2021163455A1 publication Critical patent/WO2021163455A1/fr
Priority to US17/887,341 priority Critical patent/US20220378888A1/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005—Wall or coating material
    • A61K9/5063—Compounds of unknown constitution, e.g. material from plants or animals
    • A61K9/5068—Cell membranes or bacterial membranes enclosing drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4615—Dendritic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4622—Antigen presenting cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/46449—Melanoma antigens
    • A61K39/464492—Glycoprotein 100 [Gp100]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6923—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being an inorganic particle, e.g. ceramic particles, silica particles, ferrite or synsorb
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
    • B01J13/02—Making microcapsules or microballoons
    • B01J13/06—Making microcapsules or microballoons by phase separation
    • B01J13/10—Complex coacervation, i.e. interaction of oppositely charged particles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen

Definitions

  • the present subject matter relates to a method for synthesizing cell membrane-biomimetic therapeutics, and particularly, to a method for synthesizing cell membrane-biomimetic therapeutics using flash nanocomplexation.
  • Biomimetic strategies are useful for designing therapeutic delivery systems that can negotiate biological barriers. Size reduction, colloidal stability, particle protection, and enhanced permeability and retention (EPR) properties in one or more dimensions are practical considerations in preparing nanoparticles for efficient diagnostic and therapeutic applications.
  • EPR enhanced permeability and retention
  • Cell membrane-coating of therapeutic nanoparticles is a promising biomimetic strategy.
  • Cell membrane coating technology integrates the biological features of cell membranes with the functional versatility of nanomaterials. Production involves coating synthetic nanoparticle backbone materials with a naturally-derived cell membrane layer to form a biomimicking ensemble.
  • These nano therapeutics have shown advantageous physical properties such as improved stability and longer circulation times, and intrinsic functionalities inherited from the donor cell source such as toxin neutralization, homologous targeting, and immune invasion.
  • producing regulatory agency-approved cell membrane-coated nanomaterials requires a high level of manufacturing sophistication.
  • Conventional approaches to fabricating cell membrane-coated nanomaterials rely on two main strategies: extrusion and sonication.
  • a method for synthesizing a cell membrane-loaded particle can include coating core paticles with cell membrane materials using flash nanocomplexation (FNC).
  • FNC is a turbulent mixing and self-assembly method that can produce cell membrane-coated particles in a reproducible and scalable manner.
  • the FNC-produced cell membrane-coated particles demonstrate lower aggregation, polydispersity, and zeta potential, than particles prepared by conventional coating methods, such as conventional bulk-sonication.
  • the present method achieves more complete and homogeneous coating than conventional bulk-sonication methods.
  • FNC cell membrane coating is effective even on cationic particles, which cannot be achieved using sonication methods.
  • the FNC-fabricated nanovaccines demonstrate better performance on lymph node targeting, DC antigen presentation, T cell immune-activation, and prophylactic and therapeutic efficacy in melanoma when combined with anti-CTLA-4. Accordingly, FNC represents a universal, robust, and scalable tool that can be used for the manufacturing of cell membrane-based biomimetic nanomedicine.
  • a method of using flash nanocomplexation to prepare a cell membrane- loaded particle can include loading a cell membrane material and a core particle into a confined mixing cavity and turbulent mixing of the cell membrane material and the core particle in the mixing cavity to provide the cell membrane-cloaked particles.
  • the turbulent mixing achieves a turbulent intershearing flow in the confined cavity.
  • the confined cavity includes a multi-inlet vortex mixer.
  • a flow rate in the multi inlet vortex mixer ranges from about 5 mL/min to about 40 mL/min.
  • Figs. 1A-1F depict A) a schematic illustration of FNC cell membrane coating; B) a comparison of FNC and bulk sonication methods on PDI and stability of membrane-coated nanoparticles; C) characterization of membrane-coated MSNs using different membrane-to-MSN ratios in terms of size and PDI; D) characterization of membrane-coated MSNs using different membrane-to-MSN ratios in terms of size Zeta potential; E) images of multi-inlet vortex mixer (MIVM) and vials containing total of 40 mF B16-F10 membrane-coated MSNs at 0.5 mg/mF as well as their lyophilized product; F) Total flow rate and production rate for cell membrane-coated MSN using FNC at different Reynolds numbers.
  • MIVM multi-inlet vortex mixer
  • Fig. 2 depicts SEM images of bare cores and cell membrane-coated particles produced using FNC.
  • Fig. 3 depicts TEM images of bare cores and cell membrane-coated particles produced using FNC
  • Figs. 4A-4D depict A) size and PDI of raw membrane-coated MSN-Se-Se NPs; B)size and PDI of of raw membrane-coated MSN-Se-Se-NFb NPs; C) size and Zeta of raw membrane-coated MSN-Se-Se NPs; and D) size and Zeta potential of raw membrane-coated MSN-Se-Se-NFb NPs, produced using bulk sonication or FNC methods.
  • Figs. 5A-5D depict A) size and PDI of MCF membrane- coated PFGA NPs; B) size and PDI of HepG2 membrane-coated PEI-plasmid NPs, produced by bulk sonication or FNC methods; C) size and Zeta potential of MCF membrane- coated PFGA NPs; and D) Zeta potential of HepG2 membrane-coated PEI-plasmid NPs, produced by bulk sonication or FNC methods.
  • Fig. 8 depicts SDS-PAGE protein analysis of MSN-CpG@CM produced using bulk sonication or FNC methods.
  • Fig. 9 depicts CpG release behavior of MSN-CpG@CM produced using FNC in IX PBS or 5xl0 3 M GSH or lxlO 4 M H 2 0 2 for 48 h.
  • MSN- CpG group Fluorescence imaging of popliteal lymph node at indicated time points after footpad injection of free CpG, naked MSN-CpG, or MSN-CpG@CM produced using bulk sonication or FNC methods; D) Quantitation of fluorescence intensity from Cy5.5-labeled CpG in the popliteal lymph node; and E) Uptake of Cy5.5-labeled MSN-CpG@CM by DCs and macrophages in the lymph node at 24 h after injection.
  • Figs. 13A-13D relate to APCs were incubated with nanovaccines or various control formulations and depict A) Quantification of DC maturation markers CD40, CD80, CD86 in vitro;
  • Data represent mean ⁇ SD ( /; ⁇ ().05 vs. CpG group, # p ⁇ 0.05 vs. MSN-CpG group, & p ⁇ 0.05 vs. bulk MSN-CpG@CM group).
  • Figs. 15A-15B depict secretion of A) IL-6 in DCs isolated from popliteal lymph nodes after vaccination with nanovaccines or control formulations; and B) IL-12 in DCs isolated from popliteal lymph nodes after vaccination with nanovaccines or control formulations.
  • a method for synthesizing cell membrane-cloaked particles can include coating core paticles with cell membrane materials using flash nanocomplexation (FNC).
  • FNC is a turbulent mixing and self-assembly method that can produce cell membrane-coated nano therapeutics in a reproducible and scalable manner.
  • FNC can induce electrostatic interactions which rapidly homogenize the charged core paticles with the negatively charged cell membrane materials to achieve a uniform coating of the core paticles.
  • the turbulent mixing generally involves continuous rapid mixing in a confined space and can be associated with a Reynolds number that is larger than 1600. The continous mixing achieves homogeneity and consistency of the product particles.
  • flash self-assembly provides better control of particle size and better reproducibility, scalability, and throughput capacity.
  • FNC is a kinetically controlled mixing process which exploits polyelectrolyte complexation-induced phase separation.
  • the nanocomposites can undergo self-assembly via physical interactions such as electrostatic interactions and hydrogen bonding, and are formed within milliseconds or microseconds in flash mixers.
  • the fluid dynamics of the flash mixers can be turbulent. In other words, the interaction of the liquid solutions can be robust within the mixer for better flow convection, allowing rapid, homogenous, and effective mixing for reactions.
  • the method can include loading a cell membrane material and a core particle into a confined mixing cavity and turbulent mixing of the cell membrane material and the core particle in the cavity to provide the cell membrane-cloaked particles.
  • the turbulent mixing can achieve a turbulent intershearing flow in the confined cavity.
  • Turbulent flow refers to a high dynamic flow (e.g., non-laminar flow) that renders a mixing profile with a high Reynold number.
  • One of the characteristics of the turbulent flow is an intershearing flow or turbulent intershearing flow which facilitates efficient mixing and diffusion for achieving homogeneous mixing of materials.
  • the method can provide coated particles having a coating thickness ranging from about 5 nm to about 20 nm.
  • the present method can be used to coat positively charged nanoparticles.
  • the flash-based cell membrane coating is a superior biomimetic preparation platform to standardize the membrane-coating protocol and to meet clinical translation requirements.
  • the method can be used for synthesizing vaccines, drug and gene delivery, as well as a wide range of other applications.
  • the core paticles can include at least one of nanoparticles and microparticles.
  • the core paticles can include, for example, silica particles, biodegradable polymer particles, DNA-polymer polyplex particles, and chemotherapeutic nanocrystals.
  • the polymer can include, for example, poly(lactic-co-glycolic acid) (PLGA) and polyethyleneimine (PEI)-plasmid.
  • PLGA poly(lactic-co-glycolic acid)
  • PEI polyethyleneimine
  • the core particles range in size from about 50 nm to about 2 pm with surface charge of the core particles varying from about -50 mV to about +50 mV.
  • the core particles can be loaded with an adjuvant.
  • the cell membrane coating materials can be from any suitable cell line.
  • the cell membrane coating materials can include cell membrane fragments obtained from cancer cells, non-immune cells, and immune cells.
  • Exemplary cell lines from which the cell membrane fragments can be obtained include, for example, CaCo-2, HepG2, MCF-7, RAW 264.7, HEK, HeLa, HITC, B16- F10, RBC, MSC.
  • the cell membrane material can include a tumor-associated antigen.
  • An embodiment of the present teachings is directed to a biomimetic vaccine comprising the cell membrane-cloaked particles prepared according to the present methods.
  • the biomimetic vaccine can including a core particle cloaked with a cell membrane material including a tumor- associated antigen.
  • the core particle can be loaded with an adjuvant.
  • the core particle comprises a mesoprous silica nanoparticle (MSN).
  • the adjuvant is CpG.
  • the cell membrane material and the core paticles can be loaded in a confined mixing cavity.
  • the confined mixing cavity can include a multi-inlet vortex mixer (MIVM).
  • the core paticles can be selected from mesoporous silica nanoparticles (MSNs) and silica dioxide microparticles.
  • the MSNs can be modified with an amine group to endow the MSNs with a positive surface charge.
  • the MIVM comprises 4 inlets. Turbulent mixing of the cell membrane material and the core paticles can be performed to achieve a flow rate in each inlet ranging from about 5 mL/min to about 40 mL/min.
  • the mass ratio of the cell membrane to core particle can range from about 0.1 to about 100 for coating optimization.
  • the cell membrane material and the core paticles can be mixed at a high Reynold number, e.g., typically larger than 1600, which constitutes highly turbulent mixing, in the confined mixing cavity at room temperature to achieve a well-controlled cell membrane cloaked particle or coated particle.
  • the coated particle can have a coating thickness ranging from about 5 nm to about 20 nm, depending on the flow rate and mass ratio used, and the polydispersity can be lower than 0.2.
  • the coated particles can have a relatively high colloidal stability.
  • FNC exploits the dynamic mixing of nanocomposites that undergo self-assembly via physical forces such as electrostatic interactions, whereby charged nanomaterials assemble to form nanoparticles (NPs) or to modify a NP or microparticle surface.
  • FNC can be used for mixing the cell membrane fragments and synthetic backbone materials for the robust and scalable production of cell membrane-coated paricles.
  • Uniform coated nanoparticles can be fabricated using FNC by optimizing the cell membrane/NP mixing ratio, flow rate, and composition. For example, the flow rate and mass ratios of the different mixes can be varied.
  • a significant driving force of the present method is the electrostatic interactions induced by FNC.
  • the electrostatic interactions rapidly homogenize the negatively charged cell membrane with the disparate charged nanoparticle core. Coating of the particle core is achieved while the cell membrane fragments self-assemble and land onto the outer surface of the particle uniformly. It should be noted that homogeneous cell membrane coating on a positively charged nanoparticle surface has not previously been reported.
  • the present method can standardize the otherwise unpredictable process of membrane coating of drug nanoparticles, increasing the reliability of cell-targeted drug delivery.
  • the potency of flash-based membrane coating (FNC) method was compared with bulk sonication, a conventional method for cell membrane coating.
  • the FNC- produced cell membrane-coated nanoparticles demonstrate lower aggregation, polydispersity, and zeta potential, than nanoparticles prepared by bulk-sonication.
  • Mesoporous silica nanoparticles (MSN) were modified with an amine group to endow the MSNs a positive surface charge.
  • FNC-based cell-membrane coating of positively charged MSNs has a wider coating ratio range (membrane to core) than the bulk-sonication method.
  • the present method achieves more complete and homogeneous coating than conventional bulk-sonication methods.
  • turbulent mixing of the cell membrane fragments and the core paticles can be conducted in a mixing microchamber or other confined cavity.
  • a multi-inlet vortex mixer MIVM
  • the inlet jets of the MIVM can produce kinetic energy which can transport cell membrane fragments and synthetic backbone materials into regions of small turbulent eddies and intershearing layers for better flow convection and hence faster coating.
  • the mixing ratio and flow rate can then be optimized to produce a uniform coating.
  • the turbulent mixing can have a Reynold number larger than 1600.
  • the cell membrane fragments can be evenly distributed around the particle for homogeneous coating because the mixing time ( t mixing ), during which cell membrane fragments and core particles are mixed homogenously, can be much shorter than the interacting time (i coatin g) between the cell membrane fragments and the core particles. Since the cell membrane material and nanoparticles are introduced to the flow chamber by different FNC inlets, the cell membrane fragments can be distributed around the nanoparticle evenly before they electrostatically interact with the nanoparticle surface.
  • the method can be used for coating a variety of core particles with cell membranes.
  • the method can be used to coat cationic particles, for example.
  • the method can be used to prepare nanovaccines.
  • the method can produce cancer nanovaccines, such as B16-F10 cancer cell membrane-coated mesoporous silica nanoparticles (MSNs) loaded with the adjuvant CpG.
  • MSNs cancer cell membrane-coated mesoporous silica nanoparticles
  • the FNC cell membrane coating process relies on electrostatic interactions between cell membrane fragments and core materials to form “right- side-out” membrane-coated products.
  • the “right- side-out” orientation refers to an orientation in which the membrane-bound proteins are still exposed on the outside after membrane coating.
  • the mixing time ( ⁇ mixing ) within which cell membrane fragments and backbone materials are mixed homogenously, was much larger than the interacting time (i coatin g).
  • Fig. 1A shows coating outcomes of both FNC and sonication approaches for coating nanoparticles using different nanoparticle core materials and cell membrane types.
  • PLGA poly(lactic-co-glycolic acid)
  • PEI polyethyleneimine
  • silica particles were used to obtain the cell membrane coating materials.
  • the core-shell structure of the resulting cell membrane-coated particles was confirmed using electron microscopy (Figs. 2 and 3). Increases in particle size and surface charge were measured using dynamic light scattering (DLS) (Figs. 4A-4D and 5A-5D).
  • Fig. IB shows a plot of size change of various particles immediately after coating versus change at two weeks after coating and sitting in water. A remarkable difference in membrane coating homogeneity and in particle stability between the products of the two methods was demonstrated. Across a spectrum of NPs of cell membranes, FNC products showed a smaller size change and better particle colloidal stability than the bulk- sonication products. Specifically, for many of the MSN subtypes and PLGA nanoparticles, the aggregation seen at day 14 was significantly reduced. The lower polydispersity index (PDI) for FNC products immediately after coating demonstrated a more complete coating. In the sonication method, ultrasound wave-energy pulverizes the cell membrane structure, and membrane fragments re-assemble around the nanoparticle backbone.
  • PDI polydispersity index
  • Such coating protocol is not standardized for bulk containers because the sonication power-frequency is not balanced or optimized, and the quantity of cell membrane charge over the backbone surface is not well-controlled. While electrostatic interactions are the driving force in both approaches, the FNC method achieves ultra-fast and homogeneous coating by using turbulent mixing, including turbulent intershearing flow in the microchamber. This dynamic mixing is more effective than sonication in breaking cell membranes into small fragments and mixing the components to achieve even coating.
  • FNC products showed excellent dispersion (low PDI) at nearly all membrane/MSN ratios (Fig. 1C), while sonication products showed significantly higher PDI values.
  • FNC also yielded better nanoparticle charge conversion than sonication, which suggests a more complete cell- membrane coating (Fig. ID).
  • the surface charge of a completely coated nanoparticle resembles the intrinsic charge of cell-membrane vesicles, whereas incomplete coating partially reveals the charge of nanoparticles and neutralizes the zeta potential.
  • the membrane-coated MSNs ⁇ 200 nm diameter also showed better colloidal stability in serum-containing solutions when produced with FNC than with sonication (Figs. 6A-B).
  • Cancer vaccines can be created by combining tumor-associated antigens and immune-activating adjuvants.
  • the presentation of tumor-associated antigens on cancer cell membrane-coated backbone materials together with delivery of adjuvants such as CpG can generate tumor- specific immune responses and lymph node targeting.
  • the present inventors previously fabricated multiple stimuli-responsive and biodegradable diselenide-bridged MSNs for efficient delivery of biomacromolecules for cancer therapy.
  • a biomimetic vaccine (MSN-CpG@CM) was synthesized by coating large-pore MSNs loaded with the adjuvant CpG with cancer cell membrane fragments containing tumor- specific antigens (Fig.
  • CpG 1826 was encapsulated in amine-modified MSNs (MSN-NFb) for maximum loading.
  • MSN-NFb amine-modified MSNs
  • B16-F10 mouse melanoma cell membranes were selected for the coating.
  • the FNC and bulk mixing/sonication approaches were systematically compared to determine whether they were capable of scalable production of these biomimetic cancer vaccines, and their therapeutic efficacy was evaluated in vitro and in vivo.
  • CpG-loaded MSNs were coated with B16-F10 cell membrane fragments using the FNC platform with a turbulent MIVM micromixer and using bulk sonication.
  • a membrane-to-NP mass ratio of 2:1 was selected since this value is often reported as the optimal ratio for cell membrane coating.
  • the surface morphologies of the CpG-loaded MSNs before coating (MSN-CpG) and after coating (MSN-CpG@CM) using the two methods are shown in TEM images (Fig. 7B).
  • the tumor- associated antigen gplOO is specific for targeting melanoma in drug and vaccines.
  • the presence of gplOO in the membrane coating of the MSN-CpG@CM particles was confirmed by Western blot (Fig. 7C).
  • MSN-CpG@CM at ⁇ 50 pg/mL showed no significant cytotoxicity using two types of antigen-presenting cells (APCs) (Figs. 10A- 10B).
  • a high degree of intracellular colocalization of CpG-loaded MSNs and cancer cell membrane proteins were observed in endosomes/lysosomes after 3 h of uptake (Figs. 11 A-l IE), further verifying the structural integrity and stability of the MSN-CpG@CM.
  • the uptake of MSN-CpG@CM by bone marrow-derived dendritic cells (BMDCs) was then investigated (Fig. 11B).
  • MSN-CpG@CM prepared using either FNC or bulk sonication
  • MSN-CpG@CM were then injected into mice via the foot pad, and nanovaccines were observed in the popliteal lymph node after 1 h of administration.
  • the fluorescence signal from dye-labelled CpG peaked at 12 h after injection, and started to decrease at 24 h (Fig. 11C). Quantification of mean fluorescence intensity of free CpG, naked MSN-CpG, and MSN-CpG@CM in the lymph node confirmed this observation (Fig. 11D).
  • the immunostimulatory effect of MSN-CpG@CM was characterized by assessing DC maturation and the generation of antigen- specific T cells.
  • DC maturation was assessed by measuring the expression of the costimulatory markers CD80, CD40, and CD86.
  • the secretion of TNF-a, IL-6, and IL-12 from APCs were also determined in vitro (Figs. 13A-13D).
  • CpG alone and MSN-CpG induced less potent DC maturation than MSN-CpG@CM (Fig. 14A).
  • MSN-CpG@CM produced using FNC induced greater DC maturation and secretion of IL-6 and IL-12 than MSN-CpG@CM produced using sonication (Fig.
  • APCs responses, specific immune activation, and prophylactic tumor growth inhibition in vivo were evaluated using a 16-F10 murine model (Figs. 14C-14D). Mice were vaccinated using different nanoformulations and tumor growth was monitored for up to 40 days. MSN-CpG and free CpG had no significant protective benefit, consistent with previous studies; both treatments showed a median survival of 29 d, similar to the median 26.5 d survival for the negative control. Both FNC- and sonication-produced MSN-CpG@CM groups showed tumor growth inhibition, but the FNC-produced vaccine had a much greater inhibitory effect and longer survival (Figs. 16A- 16B).
  • Performance of the MSN-CpG@CM was assessd with and without the immune checkpoint blocking antibody anti-CTLA-4. Without anti-CTLA-4, the median survival was extended from 18 d for the blank control group to 34 d for the bulk sonication MSN-CpG@CM group and 38 d for the FNC MSN-CpG@CM group (Figs. 14C and 14E). With anti-CTLA-4, the median survival was over 150 days for both FNC and sonication MSN-CpG@CM groups, indicating that combined immunotherapy produced synergistic antitumor effects. The combined therapy using FNC- produced nanovaccines with anti-CTLA-4 had the strongest antitumor effect (Figs. 17A-17B).
  • the present methods provide a nanoformulation platform for fabricating diverse cell membrane-based biomimetic NPs in a facile, reproducible, and scalable manner.
  • the FNC platform leverages dynamic turbulent mixing to homogeneously blend and uniformly distribute cell membrane fragments around NP surfaces.
  • FNC can be used to coat both negatively- and positively-charged particles with cell membranes.
  • the FNC method may enable standardization of the cell-membrane coating process for clinical translation.
  • FNC-produced MSNs loaded with CpG adjuvant and coated with a cancer cell membrane exhibited enhanced accumulation in lymph nodes and immune activation, and greater tumor growth inhibition alone and in combination treatment with the immune checkpoint-blocking antibody anti-CTLA-4 in an in vivo melanoma model.
  • High-throughput manufacturing of nanomedicine can pose a challenge for clinical and industrial translation.
  • the cell membrane-coating method described herein addresses this challenge.
  • the advantages of FNC include (1) automation, using an easy-to-transfer protocol; (2) reproducibility, reducing batch-to- batch variation; (3) user-friendliness, obviating training requirement, and (4) scalable manufacturing, facilitating clinical and industrial translation.
  • Tetraethyl orthosilicate TEOS
  • BTESPT bis[3-(triethoxysilyl)propyl]tetrasulfide
  • CP g- chloropropyl trimethoxysilane
  • APTES 3-aminopropyltriethoxysilane
  • CAT etyltrimethylammonium tosylate
  • TEAH3 triethanolamine
  • TAA triethanolamine
  • FITC fluorescein isothiocyanate
  • Hoechst 33343 bisbenzimide H-33343 trihydrochloride was purchased from VWR. LysoTracker Red DND-99 and Vybrant DiD Cell-Labeling Solution (V22887) were purchased from Thermo Fisher Scientific. ODN 1826-TLR9 ligand and ODN 1926 FITC were purchased from InvivoGen.
  • Anionic and cationic MSN-Si with an average NP diameter of 80-100 nm
  • anionic and cation MSN-Se with an average NP diameter of 80-100 nm
  • S1O2 microparticles with sizes of 1 pm and 2 pm
  • GFP plasmid-PEI NPs and PLGA NPs were selected for membrane coating. All types of MSN (e.g., Si-Si and Se-Se, disulfide and diselenide bridged MSN, respectively) with different pore sizes were synthesized in the lab using previously described methods.
  • CpG-MSN solution was placed on a shaker with 200 rpm. At each timepoint, the solution was centrifuged and the supernatant was analyzed by UV- Vis.
  • PLGA NPs were prepared by flash nanoprecipitation method as previously reported using a two-inlet confine impingement jet mixer (CIJ).
  • GFP-PEI nano-polyplexes were fabricated as previously described.
  • CIJ mixer was designed according to literature and fabricated in Columbia University Biomedical Engineering machine shop.
  • a four-stream multi-inlet vortex mixer (MIVM) was manufactured according to the literature.
  • FNC-based cell membrane coating was achieved in the manufactured four-stream MIVM.
  • B16-F10 cell membrane to MSNs ratio of 0.5 and 1 were prepared.
  • Cell membrane and CpG-MSN were introduced into the MIVM respectively.
  • a total of 120 mF/min flow rate was applied to prepare membrane-coated nanoparticles.
  • Infusion/withdrawal PHD UFTRA 4400 pumps were obtained from Harvard Apparatus.
  • MIVM multi-inlet vortex mixer
  • CaCo-2, HepG2, HEK 293 cell-membranes were exploited to coat MSNs (small pore), MSNs (big pore) and silica dioxide microparticles respectively, with the membrane/particle mass ratio of 1.
  • CaCo-2 membrane was used to coat PEI-DNA nanocomplexes with the membrane/particle mass ratio of 1.
  • RAW264.7 and B 16-F10 membrane were applied with different membrane/MSN mass ratios.
  • Four different operators without sufficient training on cell-membrane coating performed the MSNs coating experiment comparing the FNC to the bulk sonication method.
  • the flow rate in each inlet was maintained within a range of 5-40 mL/min.
  • the mass ratio of the cell membrane to nanoparticle varied from 0.1 to 100 for coating optimization. Coating was achieved by mixing the membrane and “nano-core”under high fluidic dynamic profile in the confined mixing cavity at room temperature.
  • the typical coating thickness ranged from 5 to 20 nm, depending on the flow rate and mass ratio used, and the polydispersity was basically lower than 0.2, suggesting relatively high colloidal stability.
  • the resulting micelle was stable in the serum-containing environment, such as complete cell culture medium.
  • B 16-F10 membrane was used for all in vitro and in vivo studies with the initial final-product concentration of 0.5 mg/mL. The efflux was collected and allowed to settle before further coating characterization.
  • NPs coated using the bulk sonication method equal volumes of cell membrane vesicles and core paticles were mixed, pipetted, and sonicated in 15 mL Falcon tubes in a Branson Ultrasonic Bath sonicator at 42 kHz and 100 W for 2 min. The surface zeta potential of naked and membrane-coated particles was examined by DLS using a Malvern Zetasizer.
  • PLGA NPs DLS was used to compare the size difference of bare PLGA NPs that were fabricated using double-emulsion method to the ones prepared using FNP
  • the size and zeta potential of MCF membrane-coated PLGA NPs using bulk- sonication and FNC were also evaluated by DLS.
  • To test the stability of naked and membrane-coated MSNs particles were stored for two weeks and measured by DLS every other day. Specifically, MSN-CpG@CM NPs were tested in 10% serum- containing media for the two-week stability assessment. The NPs solution concentration was 100 pg/mL.
  • TEM characterization samples were prepared and dried onto a carbon-coated copper grid.
  • Membrane-coated PLGA NPs were stained with uranyl acetate before TEM imaging. Identification of gplOO tumor antigen was performed by Western blotting.
  • B16-F10 mouse melanoma cells (CRL-6457; American Type Culture Collection), RAW 264.7 mouse macrophage cells (TIB-711; American Type Culture Collection), HepG2 human liver cancer cells (HB-8065; American Type Culture Collection), Caco-2 human epithelial colorectal cancer cells (HTB-37; American Type Culture Collection), HCT-116 human colon cancer cells (CCL-247; American Type Culture Collection), and HEK 293 human embryonic 5 kidney cells (CRL-1573) were cultured for cell membrane derivation. Cells were cultured in DMEM media with 10% fetal bovine serum (Gibco) and 100 U penicillin-streptomycin.
  • DMEM media 10% fetal bovine serum (Gibco) and 100 U penicillin-streptomycin.
  • BMDCs BMDCs
  • Healthy mice were euthanized using carbon dioxide asphyxiation followed by cervical dislocation. Both femurs were dissected, cleaned in 75% ethanol, and cut on both ends. Bone marrow was then flushed out of the bone with a 1 mL sterile syringe using warm DMEM media including 10% fetal bovine serum (Gibco) and 100 U penicillin- streptomycin.
  • BMDC growth media including the basal media further supplemented with 20 ng/mL granulocyte/macrophage-colony stimulating factor (GM-CSF; Protech), to a concentration of lxlO 6 cells/mL, and plated into petri plates at 2xl0 6 cells per plate. Media were half-changed every two days.
  • GM-CSF granulocyte/macrophage-colony stimulating factor
  • the cytotoxicity of MSN, MSN-CpG and MSN-CpG@CMs in the RAW264.7 or BMDC were assessed using an MTT assay.
  • BMDCs were collected on day 5 and plated into 24-well suspension plates. FAM-labeled CpG, MSN-CpG and MSN-CpG@CMs were added at an equivalent CpG concentration of 5 pg/mL. After 3 h incubation, the cells were washed and stained with DAPI and LysoTracker Red. 15 min later, cells were imaged by using a laser scanning confocal microscopy (CLSM). For flow cytometry, cells were collected, washed twice in PBS, and resuspended in 200 pF of 10% PBS. The cell suspension was analyzed using BD Accuri C6 plus flow cytometer. Collected data were analyzed by FlowJo software.
  • CLSM laser scanning confocal microscopy
  • BMDCs were collected on day 5, and 3xl0 6 BMDCs were plated into 6-well suspension plates in BMDC growth media. Cells were pulsed with materials for 12 h at 5 pg/mF CpG, then washed twice with fresh media. After an additional 48 h of culture, cell supernatants were collected and cytokine content was analyzed using IF-6 and IF- 12 EFISA kits. The cells were then collected and washed twice. Cells were stained with FITC-conjugated anti-mouse CD 11c and APC-conjugated anti-mouse CD40, CD80 or CD86.
  • Appropriate dye-labeled antibody isotypes (Biolegend) were used for gating purposes with cells from an untreated lymph node. Data were collected using a BD FACSCelesta flow cytometer and analyzed using FlowJo software. RAW264.7 cells were plated into 6-well suspension plates at 5x10 s cells/well and pulsed with materials for 24 h at 5 pg/mL CpG, then cell supernatants were collected and cytokine content was analyzed using TNF-a ELISA kits.
  • mice All animals received care in compliance with the guidelines outlined in the Guide for the Care and Use of Laboratory Animals, and the procedures were approved by the South China University of Technology Animal Care and Use Committee.
  • Female C57BL/6J mice were obtained at 6-10 weeks old from Hunan SJA Laboratory Animal Co., LTD.
  • Dendritic cell activation following immunization with CpG, MSN-CpG, and MSN- CpG@CMs was determined by testing DC maturation and lymph node cytokine secretion.
  • 20 pL of each material was injected into the hock.
  • the popliteal lymph nodes of all treated mice were collected into 500 pL dissociation buffer and manually dissociated.
  • Cells were stained using PE antimouse CDllc with either APC-conjugated antimouse CD40 (124611; Biolegend), CD80 (104713; Biolegend), or CD86 (105011; Biolegend).
  • lymph node-derived single cell suspensions were plated with 500 pL of BMDC growth media in 24-well tissue culture plates. After 48 h, supernatant was collected and analyzed for cytokine content using IL-6 and IL-12 ELISA kits.
  • C57BL/6J mice were vaccinated subcutaneously with 20 pL of the different materials in each hock on days 0, 2, and 4. On day 10, spleens were collected and processed into single cell suspensions. After red blood cells lysis, 5xl0 6 splenocytes were plated into 6-well suspension plates and pulsed with 1 pg/mL of mouse gplOO peptide with sequence EGSRNQDWL in BMDC growth media. After 7 days, cells were collected, washed in PBS, and stained with APC-conjugated anti-mouse 8 CD8a and phycoerythrin (PE)- labeled H-2Db gplOO tetramer. Data were collected using a BD FACSCelesta flow cytometer and analyzed using FlowJo software.
  • PE phycoerythrin
  • mice were vaccinated with 100 pL of the different materials at 0.1 mg/mL of CpG or equivalent, on days -21, -14, and -7.
  • the checkpoint blockade cocktail consisting of 100 pg anti-CTLA4 (BP0164; BioXCell) was administered intraperitoneally on the same days. Tumors were measured every other day and the experimental endpoint was defined as either death or tumor size greater than 2000 mm 2 .

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Epidemiology (AREA)
  • Immunology (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Organic Chemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Oncology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Molecular Biology (AREA)
  • Dispersion Chemistry (AREA)
  • Biophysics (AREA)
  • Zoology (AREA)
  • Virology (AREA)
  • Ceramic Engineering (AREA)
  • Inorganic Chemistry (AREA)
  • Botany (AREA)
  • Biochemistry (AREA)
  • Medicinal Preparation (AREA)

Abstract

L'invention concerne un procédé de synthèse de produits nanothérapeutiques biomimétiques de membrane cellulaire qui peut comprendre le revêtement de particules de noyau avec des matériaux de membrane cellulaire à l'aide de nanocomplexation flash (FNC). Le FNC est un procédé de mélange turbulent et d'auto-assemblage qui peut produire des produits nanothérapeutiques revêtus de membrane cellulaire d'une manière reproductible et évolutive. Les particules revêtues de membrane cellulaire produites par FNC démontrent une agrégation, une polydispersité et un potentiel zêta inférieurs à ceux des nanoparticules préparées par des procédés de revêtement classiques, tels que la sonication en masse classique. Ainsi, le présent procédé permet d'obtenir un revêtement plus complet, homogène et contrôlable que les procédés classiques de sonication en masse.
PCT/US2021/017823 2020-02-14 2021-02-12 Technologie de revêtement de membrane cellulaire évolutive et facile à la fois pour des particules chargées positivement et négativement WO2021163455A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US17/887,341 US20220378888A1 (en) 2020-02-14 2022-08-12 Scalable and facile cell-membrane-coating technology for both positively and negatively charged particles

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202062976865P 2020-02-14 2020-02-14
US62/976,865 2020-02-14

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US17/887,341 Continuation US20220378888A1 (en) 2020-02-14 2022-08-12 Scalable and facile cell-membrane-coating technology for both positively and negatively charged particles

Publications (1)

Publication Number Publication Date
WO2021163455A1 true WO2021163455A1 (fr) 2021-08-19

Family

ID=77292701

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2021/017823 WO2021163455A1 (fr) 2020-02-14 2021-02-12 Technologie de revêtement de membrane cellulaire évolutive et facile à la fois pour des particules chargées positivement et négativement

Country Status (2)

Country Link
US (1) US20220378888A1 (fr)
WO (1) WO2021163455A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024137003A1 (fr) * 2022-12-20 2024-06-27 The Curators Of The University Of Missouri Procédé évolutif et continu pour préparer des nanoparticules de médicament solide pour une thérapie de maladie oculaire

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060257485A1 (en) * 2003-03-13 2006-11-16 Eugenia Kumacheva Method of producing hybrid polymer-inorganic materials
US20130337066A1 (en) * 2011-06-02 2013-12-19 The Regents Of The University Of California Membrane Encapsulated Nanoparticles and Method of Use
US20180243229A1 (en) * 2007-11-05 2018-08-30 The Trustees Of Princeton University Composite flash-precipitated nanoparticles

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060257485A1 (en) * 2003-03-13 2006-11-16 Eugenia Kumacheva Method of producing hybrid polymer-inorganic materials
US20180243229A1 (en) * 2007-11-05 2018-08-30 The Trustees Of Princeton University Composite flash-precipitated nanoparticles
US20130337066A1 (en) * 2011-06-02 2013-12-19 The Regents Of The University Of California Membrane Encapsulated Nanoparticles and Method of Use

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
HE ZHIYU, LIU ZHIJIA, TIAN HOUKUAN, HU YIZONG, LIU LIXIN, LEONG KAM W., MAO HAI-QUAN, CHEN YONGMING: "Scalable production of core-shell nanoparticles by flash nanocomplexation to enhance mucosal transport for oral delivery of insulin", NANOSCALE, vol. 10, no. 7, 25 January 2017 (2017-01-25), pages 3307 - 3319, XP055847190, ISSN: 2040-3364, DOI: 10.1039/C7NR08047F *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024137003A1 (fr) * 2022-12-20 2024-06-27 The Curators Of The University Of Missouri Procédé évolutif et continu pour préparer des nanoparticules de médicament solide pour une thérapie de maladie oculaire

Also Published As

Publication number Publication date
US20220378888A1 (en) 2022-12-01

Similar Documents

Publication Publication Date Title
Ke et al. Physical and chemical profiles of nanoparticles for lymphatic targeting
Soltani et al. Synthetic and biological vesicular nano-carriers designed for gene delivery
Xu et al. Mannan-decorated pathogen-like polymeric nanoparticles as nanovaccine carriers for eliciting superior anticancer immunity
Hu et al. A versatile and robust platform for the scalable manufacture of biomimetic nanovaccines
Li et al. Redox-sensitive and intrinsically fluorescent photoclick hyaluronic acid nanogels for traceable and targeted delivery of cytochrome c to breast tumor in mice
Phua et al. Messenger RNA (mRNA) nanoparticle tumour vaccination
Guo et al. Engineering polymer nanoparticles using cell membrane coating technology and their application in cancer treatments: Opportunities and challenges
Li et al. Nanovaccines integrating endogenous antigens and pathogenic adjuvants elicit potent antitumor immunity
Zoghi et al. Process variables and design of experiments in liposome and nanoliposome research
Zhou et al. Nano drug delivery system for tumor immunotherapy: next-generation therapeutics
Soprano et al. Biomimetic cell-derived nanocarriers in cancer research
CN111346236B (zh) 负载肿瘤抗原的聚多巴胺纳米粒子及其制备方法与应用
Wang et al. Lymph node-targeting nanovaccines for cancer immunotherapy
US20220378888A1 (en) Scalable and facile cell-membrane-coating technology for both positively and negatively charged particles
Wang et al. Delivery of nanoparticle antigens to antigen-presenting cells: from extracellular specific targeting to intracellular responsive presentation
Parupudi et al. Nanoparticle technologies: Recent state of the art and emerging opportunities
Han et al. Image-guided in situ cancer vaccination with combination of multi-functional nano-adjuvant and an irreversible electroporation technique
Wu et al. Nanovaccines for cancer immunotherapy: current knowledge and future perspectives
Pishavar et al. Aptamer-functionalized mesenchymal stem cells-derived exosomes for targeted delivery of SN38 to colon cancer cells
Desai et al. Cancer nanovaccines: nanomaterials and clinical perspectives
Shah et al. Lipid‐Based Nanoparticles
Yang et al. Polyethyleneimine-based immunoadjuvants for designing cancer vaccines
Kesharwani et al. Nanoparticle Therapeutics: Production Technologies, Types of Nanoparticles, and Regulatory Aspects
Jäger et al. pH and ROS Responsiveness of Polymersome Nanovaccines for Antigen and Adjuvant Codelivery: An In Vitro and In Vivo Comparison
Chen et al. Promoting inter-/intra-cellular process of nanomedicine through its physicochemical properties optimization

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21753296

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21753296

Country of ref document: EP

Kind code of ref document: A1